DESCRIPTION: (Applicant's Abstract) The focus of this application is on a specific Ca2+ influx pathway: a novel, plasma membrane, Ca2+ permeable, nonselective cation channel opened by the application of caffeine. The importance of the caffeine-activated channels lies in two areas: First, even though only of few of them are open at any one time, they can have a large effect on the intracellular Ca2+ concentration, yet their physiological transduction mechanism and their possible relationship to ryanodine receptors remain to be determined. Second, because of the inherent properties of the channel and our unique imaging capabilities, we can simultaneously record the unitary current and follow the fluorescence transient due to Ca2+ influx during a single opening of the channel. Therefore, it can be used to provide the relationship between fluorescence transients and the unknown Ca2+ current for such elementary events as Ca2+ sparks and puffs due to localized release of Ca2+ from intracellular stores and, by extension, the number of ryanodine or IP3 receptors associated with these events. It can also be used to study localized Ca2+ handling in the vicinity of a single channel. We will carry out our studies at first using toad stomach smooth muscle cells because more is known about the physiology of these channels in this preparation. We will also use rat cardiac cells. Our experiments will address the following questions. What is the exact relationship between a known single channel Ca2+ current and the resulting fluorescence transient? How can this relationship be used to determine the current underlying Ca2+ sparks? What are the natural cellular processes or endogenous ligands that open the channel? Are the plasma membrane caffeine-activated channels related in some way to ryanodine receptors? What Ca2+ handling events in the environment of a single channel significantly affect the resulting change in intracellular Ca2+ and Ca2+ fluorescence during a single opening of the channel? The studies outlined in this proposal should provide new insights into mechanisms involved in excitation-contraction coupling which is at the center of the skeletal, cardiac, and smooth muscle function in both normal and disease states.